Zinc is an essential element in both enzymatic and biological systems, and is physiologically the second most abundant transition metal. The inorganic physiology of intracellular zinc is poorly understood but of emerging importance in understanding a variety of human disorders and disease states. Histochemical studies of mammalian tissues including the prostate, the insulin secreting beta cells of pancreatic islets, and the dentate neurons of the hippocampus reveal patterns of Zn(II) accumulation that are disrupted in some types of prostatic cancer, diabetes and neurodegerative disorders respectively. The function of zinc in these tissues or even within compartments of single cell organisms such as S. cerevease remains controversial. 
In order to investigate the functions of such spectroscopically silent metal ions (e.g., Ca2+ and Zn+2) in biological systems, fluorescent sensor molecules that respond to a specific metal ion in the excitation or emission spectrum have shown to be useful tools. Several kinds of fluorescence probes for Zn2+ that can be used under physiological condition have been reported to date, most of these utilize “on-off” fluorescent signaling system, in which fluorescence intensity increases lineally upon increasing Zn2+ concentration. In these cases, however, the determination of the accurate Zn2+ concentration in the cells should be impossible because the fluorescence intensity depends on many factors such as the cell thickness, incubation time, illumination intensity, dye concentration, and the photobleach of dye itself.
Confocal fluorescence microscopy has proved to be a central tool in understanding calcium biology and has the potential to resolve these issues in zinc biology. On the other hand, two-photon excitation (TPE) fluorescence microscopy provides significant advantages over standard laser confocal approaches by providing deeper sectioning, less phototoxicity and selective excitation of a smaller focal volume (i.e., femptoliter), thus decreasing background fluorescence. Such advances in instrumentation could be applied to the study of zinc physiology but would require the parallel development of new zinc-specific chemical probes that operate within cells.
Ratiometric probes that exhibit a large shift in the excitation and/or emission spectrum upon binding with the cation can minimize experimental error as the fluorescence intensity ratio between the apo form and the Zn2+-bound form is dependent on only free Zn2+ concentration. Recently, two ratiometric probes for Zn2+ have been reported; however, as excitation probes, two different excitation wavelengths are needed for ratiometic cell imaging. As a result, this technique is not suitable for a confocal laser microscopy.
Such development has been an ongoing concern in the art. Protein-based zinc probes are useful in a variety of physiological experiments, but cannot be used without microinjection into each cell. Several benzofuran-based and coumazin probes have been used but only for excitation ratio imaging of a zinc loaded cell. Others in the art used 2-(2′-hydroxyphenyl)benzoxazole (HBO) as a fluorophore, which exhibits dual emission, utilizing ESIPT (Excited State Intramolecular Proton Transfer); however, intracellular analysis/imaging was not described. The search continues for a class of emission ratiometric probes for intracellular zinc, especially those with utility in two-photon fluorescence microscopy of mammalian cells.